Detailed Investigation of the Immunodominant Role of O‐Antigen Stoichiometric O‐Acetylation as Revealed by Chemical Synthesis, Immunochemistry, Solution Conformation and STD‐NMR Spectroscopy for Shigella flexneri 3a

Abstract

Shigella flexneri 3a causes bacillary dysentery. Its O-antigen has the {2)-[α-d-Glcp-(1→3)]-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-[Ac→2]-α-l-Rhap-(1→3)-[Ac→6]≈40 % -β-d-GlcpNAc-(1→} ([(E)ABAc CAc D]) repeating unit, and the non-O-acetylated equivalent defines S. flexneri X. Propyl hepta-, octa-, and decasaccharides sharing the (E')A'BAc CD(E)A sequence, and their non-O-acetylated analogues were synthesized from a fully protected BAc CD(E)A allyl glycoside. The stepwise introduction of orthogonally protected mono- and disaccharide imidate donors was followed by a two-step deprotection process. Monoclonal antibody binding to twenty-six S. flexneri types 3a and X di- to decasaccharides was studied by an inhibition enzyme-linked immunosorbent assay (ELISA) and STD-NMR spectroscopy. Epitope mapping revealed that the 2C -acetate dominated the recognition by monoclonal IgG and IgM antibodies and that the BAc CD segment was essential for binding. The glucosyl side chain contributed to a lesser extent, albeit increasingly with the chain length. Moreover, tr-NOESY analysis also showed interaction but did not reveal any meaningful conformational change upon antibody binding.

Efficient Iterative Synthesis of O‐Acetylated Tri‐ to Pentadecasaccharides Related to the Lipopolysaccharide of Shigella flexneri Type 3 a through Di‐ and Trisaccharide Glycosyl Donors

“…To identify the minimum structural requirements for recognition by homologous protective monoclonal antibodies (mAbs), our laboratory previously reported on synthetic frame‐shifted fragments of the SF3a O‐SP and on their antigenic properties . Available data revealed the presence of partially overlapping epitopes displayed over oligosaccharides larger than 1 RU . Therefore, the need to access synthetic SF3a O‐SP fragments bearing one or more RUs became imperative.…”

“…Our originally disclosed synthetic routes to oligosaccharides related to the O‐SP of SF3a were strongly inspired by our achievements in the synthesis of oligosaccharides related to the O‐SP of SF2a . In particular, a 2‐deoxy‐2‐trichloroacetamido‐4,6‐ O ‐isopropylidene‐ d ‐glucopyranose residue served as precursor to the 2‐ N ‐acetyl‐ d ‐glucosamine residue D. However, in contrast to previous observations, investigation in the SF3a series revealed the high sensitivity to reaction temperature of the 4 D ,6 D ‐ O ‐isopropylidene acetal during glycosylation at OH‐3 D of a D(E)A‐containing acceptor . Loss of the acetal moiety always took place post glycosylation, if observed.…”

“…Whereas this loss was easily circumvented by re‐acetalation of the crude glycosylation material, it impaired the robustness of the iterative process we were aiming for. Moreover, drawbacks stemming from the trichloroacetamide to acetamide conversion, resembling those outlined by others dealing with complex targets, and the demonstrated lack of stereoselectivity of the α‐ l ‐rhamnosylation reactions performed in the absence of anchimeric assistance or supposedly facilitated by an ester participating group, supported novel developments to overcome obstacles entailed by the synthesis of SF3a oligosaccharides larger than one O‐SP RU.…”

“…Whereas none of the detected intermediates comprised three chlorine atoms, dichlorinated 38 {HRMS (ESI): m / z calcd for C 105 H 171 Cl 2 N 3 O 70 Na 2 : 1354.9543 [ M +2 Na] 2+ ; found: 1354.9496 and monochlorinated 39 and/or 40 {HRMS (ESI): m / z calcd for C 105 H 172 ClN 3 O 70 Na 2 : 1337.9738 [ M +2 Na] 2+ ; found: 1337.9808 species were the major compounds. We observed previously, during the synthesis of the SF3a octasaccharide [B Ac CD(E)A] 2 ‐Pr and decasaccharide [B Ac CD(E)A] 2 ‐Pr, that monochlorinated analogues at hindered glucosamine residues were isolated in significant amounts upon final Pd/C‐mediated hydrogenolysis. Accordingly, decasaccharide 37 and pentadecasaccharides 38 , 39 , and 40 , bearing a chloroacetamide moiety at all the inner glucosamine residues, appear to be the most probable intermediates in the final deprotection process leading to propyl glycosides 2 and 3 , respectively.…”

Increasing the Affinity of an O-Antigen Polysaccharide Binding Site in Shigella Flexneri Bacteriophage Sf6 Tailspike Protein

“…Interactions between oligo-or polysaccharides as ligands to proteins or antibodies can be investigated by trNOESY NMR experiments. [38] The S. flexneri Yo ctasaccharide contains a-lrhamnosyl residues to al arge extent, like for any of the O-antigens from various S. flexneri serotypes. The methyl groups at C6 of l-rhamnosyl residues thus enable an increased flexibility of the glycans compared to contributions of other,f ully hydroxylated monosaccharide buildingb locks.…”